Insect development appears to be driven by the action of at least two hormone classes, the ecdysteroids and the juvenile hormones (JHs, juvenoids) (Riddiford, 1994, Adv. Insect Physiol., 24:213-274; Gilbert et al., 2000, Insect Biochem. Mol. Biol., 30:617-644; Thummel, 2002, Insect Biochem. Mol. Biol., 32:113-120). It appears that ecdysteroids are responsible for initiating metamorphosis, and in some insects, regulating adult fertility. In contrast, JH appears to be required for reproductive processes such as adult female vitellogenesis (Wyatt, G. R., and K. G. Davey, 1996, Adv. Insect Physiol., 26:1-15). Also, the simultaneous presence of ecdysteroids and juvenile hormone (JH) leads to larval-larval molting.
The insect ecdysteroid receptor is a heterodimer comprised of two nuclear receptors, the ecdysone receptor (EcR; Koelle et al, 1991, Cell, 4:59-77), and Ultraspiracle (USP; Oro et al, 1990, Nature, 347, 298-301; Henrich et al, 1990; Shea et al, 1990), that are stabilized by the presence of the insect molting hormone, 20-hydroxyecdysone (20E; Yao et al, 1992, Cell, 71:63-72; Yao et al, 1993, Nature, 366: 476-479; Thomas et al, 1993, Nature, 362:471-475). The heterodimer can interact with any one of several defined DNA sequence elements to regulate the transcription of target genes (Antoniewski et al, 1993, Insect Biochem. Mol. Biol., 23:105-114; Vogtli et al, 1998, Nuc. Acids Res., 10:2407-2414). Both EcR and USP appear to be required for the normal progression of premetamorphic development in Drosophila melanogaster (Hall and Thummel, 1998, Development, 125:4709-4717; Bender et al, 1997, Cell, 91:777-788; Li and Bender, 2001,; Henrich et al, 2000). Ecdysteroids represent the only endogenous class of steroid hormones known in D. melanogaster and other insects, and this single class can lead to both cell-specific and generally shared transcriptional changes (Riddiford et al, 2000, Vitam. Horm., 60:1-73; Thummel, 2002).
There may also be mammalian counterparts to the insect receptor for ectdysteroids. Thus, EcR structurally resembles the vertebrate farnesoid X-activated receptor (FXR) (Forman, B. M., et al., 1995, Cell, 81:687-695). FXR is a member of the steroid receptor family that includes receptors for glucocorticoids, estrogen, vitamins A and D, thyroid hormones, and fatty acids. Also, there is evidence to suggest that USP may be the insect orthologue of the vertebrate retinoid X receptor (RXR) (Oro, et al., 1990). Comparisons of amino acids in the FXR and EcR DNA-binding domains reveal 60% identity, and the ligand binding domains (LBD) regions of these two receptors share about 45% identity. Nevertheless, the possible functional analogy between FXR/RXR and EcR/USP has yet to be resolved. For example, it has been shown that the vertebrate RXR is activated by methoprene acid but not JHIII or methoprene (Harmon, M. A., et al., 1995, Proc. Natl. Acad. Sci. USA, 92:6157-6160). In contrast, the insect USP complexes with JHIII and methoprene but not their acid metabolites (Jones, G. et al., 1997, Proc. Natl. Acad. Sci. USA, 94:13499-13503).
The diversity of ecdysteroid responses found among developing Drosophila tissues may involve the distinct roles of the three natural isoforms of EcR-EcRA, EcRB1, and EcRB2 (Talbot et al, 1993, Cell, 73:1323-1337). Expression of these isoforms appears to be regulated by different EcR promoters (A and B forms; Sung and Robinow, 2000, Mech. Dev., 91:237-248) and alternative splicing (B1 and B2) of a single gene, such that the level and distribution of each isoform may vary among tissues (Talbot et al, 1993). Distribution of EcR isoforms in different tissues, however, has not yet been clearly correlated with isoform-specific phenotypic disruptions (Cherbas et al, 2003, Development, 130:271-284). Still, the B isoforms have been associated with larval functions (Schubiger et al, 1998, Development, 125:2053-2062). The B1 isoform is appears to be the isoform that is solely capable of mediating the ecdysteroid response in salivary gland cells (Bender et al, 1997). B2 seems to be the most efficient isoform for rescuing larval development in EcR mutants (Li and Bender, 2001). Thus, there appears to be a functional distinction between the two B isoforms. By contrast, the A isoform has been implicated in the remodelling of neurons during metamorphosis (Robinow et al, 1993, Development, 119:1251-1259). Isoform-specific mutations of EcR further reveal the distinct functional roles during development. Generally, such mutations are lethal prior to metamorphosis. For example, many, although not all, mutations of EcR coding regions shared by the three isoforms cause death during embryogenesis (Bender et al, 1997; Carney and Bender, 2000, Genetics, 154:1203-1211).
Drosophila USP appears to be essential for metamorphosis (Hall and Thummel, 1998). Interestingly, the absence of USP during the late third instar induces subtly different phenotypic effects than the absence of EcR, suggesting divergent roles for these two proteins as metamorphosis approaches (Li and Bender, 2001). An interspecial chimeric USP has further revealed distinct larval and metamorphic functions for USP (Henrich et al, 2000). Also, evidence indicates that USP performs separable repressive and inductive functions (Schubiger and Truman, 2000, Development, 127:1151-1159; Ghbeish et al, 2001, Proc. Natl. Acad. Sci., USA, 98:3867-3872; Ghbeish and McKeown, 2002, Mech. Devel., 111:89-98).
Cell culture studies have demonstrated that the individual Drosophila EcR isoforms may not be equivalent in their performance. Despite these differences, each of the isolforms appear to mediate elevated transcription levels in response to ecdysteroids (Mouillet et al, 2001, Eur. J. Biochem., 268:1811-1819). Also, each of the isoforms are potentiated by the simultaneous presence of JHIII with an activating ecdysteroid (Henrich et al, 2003, Insect Biochem. Mol. Biol., 33:1239-1247).
Thus, insect development is regulated on a variety of levels. There is a need for the development of compounds that can interrupt insect morphogenesis and growth in a highly specific manner, so as to avoid any untoward side effects. Also, there is a need to develop species-specific and/or developmental stage-specific insecticides, so that insects that are detrimental may be specifically targeting while minimizing adverse effects on beneficial species.